School of Chemical and Bioprocess Engineering, University College Dublin, Belfield, Dublin 4, Ireland.
BMC Biotechnol. 2010 Mar 22;10:25. doi: 10.1186/1472-6750-10-25.
The biopharmaceutical industry requires cell lines to have an optimal proliferation rate and a high integral viable cell number resulting in a maximum volumetric recombinant protein product titre. Nutrient feeding has been shown to boost cell number and productivity in fed-batch culture, but cell line engineering is another route one may take to increase these parameters in the bioreactor. The use of CHO-K1 cells with a c-myc plasmid allowing for over-expressing c-Myc (designated cMycCHO) gives a higher integral viable cell number. In this study the differential protein expression in cMycCHO is investigated using two-dimensional gel electrophoresis (2-DE) followed by image analysis to determine the extent of the effect c-Myc has on the cell and the proteins involved to give the new phenotype.
Over 100 proteins that were differentially expressed in cMycCHO cells were detected with high statistical confidence, of which 41 were subsequently identified by tandem mass spectrometry (LC-MS/MS). Further analysis revealed proteins involved in a variety of pathways. Some examples of changes in protein expression include: an increase in nucleolin, involved in proliferation and known to aid in stabilising anti-apoptotic protein mRNA levels, the cytoskeleton and mitochondrial morphology (vimentin), protein biosynthesis (eIF6) and energy metabolism (ATP synthetase), and a decreased regulation of all proteins, identified, involved in matrix and cell to cell adhesion.
These results indicate several proteins involved in proliferation and adhesion that could be useful for future approaches to improve proliferation and decrease adhesion of CHO cell lines which are difficult to adapt to suspension culture.
生物制药行业需要细胞系具有最佳的增殖率和较高的整体活细胞数，从而获得最大的重组蛋白产物滴度。已证明补料分批培养中的营养物补加可以提高细胞数量和产物得率，但细胞系工程是另一种可以提高生物反应器中这些参数的方法。使用带有 c-myc 质粒的 CHO-K1 细胞可以过表达 c-Myc（命名为 cMycCHO），从而获得更高的整体活细胞数。在这项研究中，使用二维凝胶电泳（2-DE）结合图像分析来研究 cMycCHO 中的差异蛋白表达，以确定 c-Myc 对细胞和涉及的蛋白的影响程度，从而赋予新的表型。
用高统计置信度检测到超过 100 种在 cMycCHO 细胞中差异表达的蛋白质，其中 41 种随后通过串联质谱（LC-MS/MS）鉴定。进一步的分析表明，这些蛋白涉及多种途径。蛋白表达变化的一些例子包括：核仁素增加，核仁素参与增殖，已知有助于稳定抗凋亡蛋白 mRNA 水平，细胞骨架和线粒体形态（波形蛋白），蛋白生物合成（eIF6）和能量代谢（ATP 合酶），以及基质和细胞间黏附所涉及的所有蛋白的调节减少。
这些结果表明，有几种与增殖和黏附有关的蛋白可能有助于未来的方法来提高 CHO 细胞系的增殖和减少黏附，CHO 细胞系难以适应悬浮培养。
